Feed formulations are required to provide animals with essential nutrients critical to growth. However, crop plants are generally rendered food sources of poor nutritional quality because they contain low proportions of several amino acids which are essential for, but cannot be synthesized by animals. For example, soybean meal in deficient in the essential sulfur-containing amino acids, methionine and cysteine, because the most abundant proteins accumulated in soybean seeds are relatively low in these amino acids. Supplementation of animal feeds to bring the levels of essential amino acids up to that required constitutes significant added expense.
For many years, researchers have attempted to improve the balance of essential amino acids in the seed proteins of important crops through breeding programs. Efforts utilizing classical breeding and mutant selection have met with limited success, probably because little variability exists in seed amino acid content. While there have been recent reports of the selection of arabidopsis mutants that overaccumulate high levels of free methionine, because free methionine represents only a small fraction of total methionine, the reported increases in free methionine translate to insignificant changes in total methionine content.
As more becomes known about seed storage proteins and the expression of the genes which encode these proteins, and as transformation systems are developed for a greater variety of plants, molecular approaches for improving the nutritional quality of seed proteins can provide alternatives to the more conventional approaches. Thus, specific amino acid levels can be enhanced in a given crop via biotechnology.
One alternative method is to express a heterologous protein of favorable amino acid composition at levels sufficient to obviate or significantly reduce feed supplementation. For this purpose, a number of seed proteins rich in sulfur amino acids have been identified. A key to good expression of such proteins involves efficient expression cassettes with seed specific promoters. Not only must the gene-controlling regions direct the synthesis of high levels of mRNA, the mRNA must be translated into stable protein.
Essential amino acids, needed for animal nutrition but often limited in crop plants, include methionine, threonine, cysteine and lysine. Attempts to increase the levels of these free amino acids by breeding, mutant selection and/or changing the composition of the storage proteins accumulated in crop plants have met with minimal success. Usually, the expression of the transgenic storage protein does not result in sufficient increase in the total seed amino acid. The phaseolin-promoted Brazil nut 2S expression cassette is an example of an effective chimeric seed-specific gene. However, even though Brazil nut protein increases the amount of total methionine and bound methionine, thereby improving nutritional value, there appears to be a threshold limitation as to the total amount of methionine that is accumulated in the seeds. The seeds remain insufficient as sources of methionine and significant methionine supplementation is required in diets utilizing the above soybeans.
An alternative to the enhancement of specific amino acid levels by altering the levels of proteins containing the desired amino acid is modification of amino acid biosynthesis. Recombinant DNA and gene transfer technologies have been applied to alter enzyme activity catalyzing key steps in the amino acid biosynthetic pathway. Glassman, U.S. Pat. No. 5,258,300; Galili, et al., European Patent Application No. 485970; (1992); incorporated herein in its entirety. However, modification of the amino acid levels in seeds is not always correlated with changes in the level of proteins that incorporate those amino acids. Burrow, et al., Mol. Gen. Genet.; Vol. 241; pp. 431-439; (1993); incorporated herein in its entirety by reference. Increases in free lysine levels in leaves and seeds have been obtained by selection for DHDPS mutants or by expressing the E. coli DHDPS in plants. However, since the level of free amino acids in seeds, in general, is only a minor fraction of the total amino acid content, these increases have been insufficient to significantly increase the total amino acid content of seed.
The lysC gene is a mutant bacterial aspartate kinase which is desensitized to feedback inhibition by lysine and threonine. Expression of this gene results in an increase in the level of lysine and threonine biosynthesis. However, expression of this gene with seed-specific expression cassettes has resulted in only a 6-7% increase in the level of total threonine or methionine in the seed. See Karchi, et al., The Plant J.; Vol. 3; pp. 721-7; (1993); incorporated herein in its entirety by reference. Thus, there is minimal impact on the nutritional value of seeds, and supplementation with essential amino acids is still required.
Based on the foregoing, there exists a need for methods of increasing the levels of essential amino acids in seeds of plants. As can be seen from the prior art, previous approaches have led to insufficient increases in the levels of both free and bound amino acids to significantly enhance the nutritional content of the feed. There exists a need to significantly increase the levels of the essential amino acids in seeds.
It is therefore an object of the present invention to provide methods for genetically modifying plants to increase the levels of essential amino acids in the seeds of such plants.
It is a further object of the present invention to provide seeds for food and/or feed with higher levels of essential amino acids than the wild type species of the same seeds.
It is a further object of the present invention to provide seeds for food and/or feed such that the level of the essential amino acids is increased, thus obviating or reducing the need for feed supplementation.